Compounds and methods for modulating neurite outgrowth

ABSTRACT

Modulating agents comprising cyclic peptides, and compositions comprising such modulating agents are provided. The cyclic peptides comprise a cadherin cell adhesion recognition sequence HAV. Methods for using such peptides and compositions for modulating and/or directing neurite outgrowth in a variety of contexts are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/996,679,filed Dec. 23, 1997, which is a continuation-in-part of U.S. Ser. No.08/893,534, filed Jul. 11, 1997, now U.S. Pat. No. 6,031,072, whichclaims the benefit of U.S. Provisional Application No. 60/021,612, filedon Jul. 12, 1996.

TECHNICAL FIELD

The present invention relates generally to methods for modulatingN-cadherin mediated processes, and more particularly to the use ofcyclic peptides comprising a cadherin cell adhesion recognition sequencefor inhibiting or enhancing cadherin-mediated neurite outgrowth.

BACKGROUND OF THE INVENTION

Nerve growth is promoted by a wide range of molecules, including thecell surface adhesion molecules (CAMs) NCAM and N-cadherin. Inparticular, N-cadherin is the predominant mediator of calcium-dependentadhesion in the nervous system. N-cadherin is a member of the classicalcadherin family of calcium-dependent CAMs (Munro et al., In. CellAdhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RGLandes Co.(Austin Tex., 1996). The classical cadherins (abbreviatedCADs) are integral membrane glycoproteins that generally promote celladhesion through homophilic interactions (a CAD on the surface of onecell binds to an identical CAD on the surface of another cell), althoughCADs also appear to be capable of forming heterotypic complexes with oneanother under certain circumstances and with lower affinity. Cadherinshave been shown to regulate epithelial, endothelial, neural and cancercell adhesion, with different CADs expressed on different cell types. N(neural)-cadherin is predominantly expressed by neural cells,endothelial cells and a variety of cancer cell types. A detaileddiscussion of the classical cadherins is provided in Munro S B et al.,1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt,ed., pp.17-34 (RG Landes Company, Austin Tex.).

The structures of the CADs are generally similar. As illustrated in FIG.1, CADs are composed of five extracellular domains (EC1-EC5), a singlehydrophobic domain (TM) that transverses the plasma membrane (PM), andtwo cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN(SEQ ID NO:8), DXD and LDRE (SEQ ID NO:9) are interspersed throughoutthe extracellular domains. The first extracellular domain (EC1) containsthe classical cadherin cell adhesion recognition (CAR) sequence, HAV(His-Ala-Val), along with flanking sequences on either side of the CARsequence that may play a role in conferring specificity. Syntheticpeptides containing the CAR sequence and antibodies directed against theCAR sequence have been shown to inhibit CAD-dependent processes (Munroet al., supra; Blaschuk et al., J. Mol. Biol. 211:679-82, 1990; Blaschuket al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell.Physiol. 156:610-18, 1993). The three-dimensional solution and crystalstructures of the EC1 domain have been determined (Overduin et al.,Science 267:386-389, 1995; Shapiro et al., Nature 374:327-337, 1995).

N-cadherin is known to promote neurite outgrowth via a homophilicbinding mechanism. N-cadherin is normally found on both the advancinggrowth cone and on cellular substrates, and the inhibition of N-cadherinfunction results in diminished neurite outgrowth. Such inhibition may bethe result of pathology or injury involving severed neuronal connectionsand/or spinal cord damage. In such cases, enhancement of N-cadherinmediated neurite outgrowth would be beneficial. However, previousattempts to promote neurite outgrowth have achieved limited success due,in part, to difficulties associated with maintaining continuous growthover a particular defined region.

Accordingly, there is a need in the art for compounds that modulateand/or direct neurite outgrowth without such disadvantages. The presentinvention fulfills this need and further provides other relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides methods for modulating cadherin-mediatedneurite outgrowth. Within one aspect, the present invention providesmethods for enhancing and/or directing neurite outgrowth, comprisingcontacting a neuron with a cell adhesion modulating agent, wherein themodulating agent enhances cadherin-mediated cell adhesion.

Within a related aspect, methods for treating spinal cord injuries in amammal are provided, comprising administering to a mammal a celladhesion modulating agent as described above, wherein the modulatingagent enhances cadherin-mediated cell adhesion.

Within the above aspects, cell adhesion modulating agents generallycomprise a cyclic peptide in which nonadjacent amino acid residues arecovalently linked to form a peptide ring, wherein the peptide ringcomprises the sequence His-Ala-Val. Within certain embodiments, thecyclic peptide has the formula:

wherein X₁, and X₂ are optional, and if present, are independentlyselected from the group consisting of amino acid residues andcombinations thereof in which the residues are linked by peptide bonds,and wherein X₁ and X₂ independently range in size from 0 to 10 residues,such that the sum of residues contained within X₁ and X₂ ranges from 1to 12; wherein Y₁ and Y₂ are independently selected from the groupconsisting of amino acid residues, and wherein a covalent bond is formedbetween residues Y₁ and Y₂; and wherein Z₁ and Z₂ are optional, and ifpresent, are independently selected from the group consisting of aminoacid residues and combinations thereof in which the residues are linkedby peptide bonds. Within certain specific embodiments Z₁ is not presentand Y₂ comprises an N-acetyl group and/or Z₂ is not present and Y₂comprises a C-terminal amide group. Linkage of Y₁ and Y₂ may be achievedvia, for example, a disulfide bond, an amide bond or a thioether bond.Certain preferred modulating agents comprise a sequence selected fromthe group consisting of N-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂(SEQ ID NO:11), N-Ac-CAHAVC-NH₂ (SEQ ID NO:12), N-Ac-CAHAVDC-NH₂ (SEQ IDNO:13), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:14), N-Ac-CRAHAVDC-NH₂ (SEQ IDNO:15), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:16), N-Ac-DHAVK-NH₂ (SEQ IDNO:17), N-Ac-KHAVE-NH₂ (SEQ ID NO:18), N-Ac-AHAVDI-NH₂ (SEQ ID NO:19)and derivatives of the foregoing sequences having one or moreC-terminal, N-terminal and/or side chain modifications. Modulatingagents may comprise multiple HAV sequences separated by a linker.Modulating agents may further be linked to one or more of a drug, asolid support, a targeting agent, a cell adhesion recognition sequencethat is bound by an adhesion molecule other than a cadherin, wherein thecell adhesion recognition sequence is separated from any HAV sequence(s)by a linker; and/or an antibody or antigen-binding fragment thereof thatspecifically binds to a cell adhesion recognition sequence bound by anadhesion molecule other than a cadherin. A modulating agent may bepresent within a pharmaceutical composition that comprises apharmaceutically acceptable carrier and, optionally, may furthercomprise a drug, a peptide comprising a cell adhesion recognitionsequence that is bound by an adhesion molecule other than a cadherin;and/or an antibody or antigen-binding fragment thereof that specificallybinds to a cell adhesion recognition sequence bound by an adhesionmolecule other than a cadherin.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.All references disclosed herein are hereby incorporated by reference intheir entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the structure of classical CADs. The fiveextracellular domains are designated EC1-EC5, the hydrophobic domainthat transverses the plasma membrane (PM) is represented by TM, and thetwo cytoplasmic domains are represented by CP1 and CP2. The calciumbinding motifs are shown by DXNDN (SEQ ID NO:8), DXD and LDRE (SEQ IDNO:9). The CAR sequence, HAV, is shown within EC1. Cytoplasmic proteinsβ-catenin (β), α-catenin (α) and α-actinin (ACT), which mediate theinteraction between CADs and microfilaments (MF) are also shown.

FIG. 2 provides the amino acid sequences of mammalian classical cadherinEC1 domains: human N-cadherin (SEQ ID NO:1), mouse N-cadherin (SEQ IDNO:2), cow N-cadherin (SEQ ID NO:3), human P-cadherin (SEQ ID NO:4),mouse P-cadherin (SEQ ID NO:5), human E-cadherin (SEQ ID NO:6) and mouseE-cadherin (SEQ ID NO:7).

FIGS. 3A-3I provide the structures of representative cyclic peptides ofthe present invention (SEQ ID NOs: 10-19,41,43,49,53,59,65,68,70 and 76;structures on the left hand side), along with similar, but inactive,structures (SEQ ID NOs: 20,53-58,60-64,66,67,69,71-75 and 77; on theright).

FIG. 4 is a histogram depicting the mean neurite length in microns forneurons grown on a monolayer of untransfected 3T3 cells (first column)or 3T3 cells transfected with cDNA encoding N-cadherin (columns 2-4). Inthe third column, the mean neurite length in the presence of therepresentative cyclic peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10) is shown.Column 4 depicts the mean neurite length in the presence of the controlpeptide N-Ac-CHGVC-NH₂ (SEQ ID NO:20).

FIG. 5 is a graph showing a dose response curve for the representativecyclic peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10) on control 3T3 cells (opencircles) and on 3T3 cells expressing N-cadherin (solid circles).

FIG. 6 is a histogram depicting the mean neurite length in microns forneurons grown in the presence (solid bars) or absence (cross-hatchedbars) of 500 μg/mL of the representative cyclic peptide N-Ac-CHAVC-NH₂(SEQ ID NO:10). In the first pair of bars, neurons were grown on amonolayer of untransfected 3T3 cells. In the remaining columns, the meanneurite length is shown for neurons cultured on 3T3 cells transfectedwith cDNA encoding N-CAM (second pair of bars), L1 (third pair of bars)or N-cadherin (fourth pair of bars).

FIG. 7 is a histogram illustrating the ability of various representativemodulating agents to inhibit neurite outgrowth. The percent inhibitionis shown for the cyclic peptide modulating agents indicated.

FIG. 8 is a graph illustrating the results of a study to assess thechronic toxicity of a representative cyclic peptide. The graph presentsthe mean body weight during the three-day treatment period (oneintraperitoneal injection per day) and the four subsequent recoverydays. Three different doses are illustrated, as indicated.

FIG. 9 is a graph illustrating the stability of a representative cyclicpeptide in mouse whole blood. The percent of the cyclic peptideremaining in the blood was assayed at various time points, as indicated.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides cell adhesion modulatingagents that are capable of modulating cadherin-mediated processes, suchas neurite outgrowth. In general, to modulate (i.e., enhance, inhibitand/or direct) neurite outgrowth, a cadherin-expressing neuron iscontacted with a cell adhesion modulating agent (also referred to hereinas a “modulating agent”) either in vivo or in vitro. A modulating agentgenerally comprises a cyclic peptide that contains the classicalcadherin cell adhesion recognition (CAR) sequence HAV (i.e.,His-Ala-Val). Such modulating agents may further comprise one or moreadditional CAR sequences and/or an antibody (or antigen-binding fragmentthereof) that specifically binds to a cadherin or other CAR sequence, asdescribed below.

CELL ADHESION MODULATING AGENTS

The term “cell adhesion modulating agent,” as used herein, refers to amolecule comprising at least one cyclic peptide that contains a cadherincell adhesion recognition (CAR) sequence, generally HAV (His-Ala-Val).The term “cyclic peptide,” as used herein, refers to a peptide or saltthereof that comprises (1) an intramolecular covalent bond between twonon-adjacent residues, forming a cyclic peptide ring and (2) at leastone cadherin CAR sequence located within the ring. The intramolecularbond may be a backbone to backbone, side-chain to backbone or side-chainto side-chain bond (i.e., terminal functional groups of a linear peptideand/or side chain functional groups of a terminal or interior residuemay be linked to achieve cyclization). Preferred intramolecular bondsinclude, but are not limited to, disulfide, amide and thioether bonds.In addition to the cadherin CAR sequence HAV, a modulating agent maycomprise additional CAR sequences, which may or may not be cadherin CARsequences, and/or antibodies or fragments thereof that specificallyrecognize a CAR sequence. Additional CAR sequences may be present withinthe cyclic peptide containing the HAV sequence, within a separate cyclicpeptide component of the modulating agent and/or in a non-cyclic portionof the modulating agent. Antibodies and antigen-binding fragmentsthereof are typically present in a non-cyclic portion of the modulatingagent.

In addition to the CAR sequence(s), cyclic peptides generally compriseat least one additional residue, such that the size of the cyclicpeptide ring ranges from 4 to about 15 residues, preferably from 5 to 10residues. Such additional residue(s) may be present on the N-terminaland/or C-terminal side of a CAR sequence, and may be derived fromsequences that flank the HAV sequence within one or more naturallyoccurring cadherins (e.g., N-cadherin) with or without amino acidsubstitutions and/or other modifications. Flanking sequences forendogenous N-, E-, P- and R-cadherin are shown in FIG. 2, and in SEQ IDNOs: 1 to 7. For modulating neurite outgrowth, such flanking sequencesare preferably derived from N-cadherin. Database accession numbers forrepresentative naturally occurring N-cadherins are as follows: humanN-cadherin M34064, mouse N-cadherin M31131 and M22556 and cow N-cadherinX53615. Alternatively, additional residues present on one or both sidesof the CAR sequence(s) may be unrelated to an endogenous sequence (e.g.,residues that facilitate cyclization).

Within certain preferred embodiments, as discussed below, relativelysmall cyclic peptides that do not contain significant sequences flankingthe HAV sequence are preferred. Such peptides may contain an N-acetylgroup and a C-amide group (e.g, the 5-residue ring N-Ac-CHAVC-NH₂ (SEQID NO:10)). Such cyclic peptides can be thought of as “master keys” thatfit into peptide binding sites of each of the different classicalcadherins, and are capable of modulating neurite outgrowth as well asadhesion of neural cells, endothelial cells, epithelial cells and/orcertain cancer cells. Small cyclic peptides may generally be used tospecifically modulate neurite outgrowth by topical administration or bysystemic administration, with or without linking a targeting agent tothe peptide, as discussed below.

Within other preferred embodiments, as noted above, a cyclic peptide maycontain sequences that flank the HAV sequence on one or both sides thatare designed to confer specificity for a function of one or morespecific cadherins (e.g., N-cadherin), resulting in tissue and/orcell-type specificity. Suitable flanking sequences for conferringspecificity include, but are not limited to, endogenous sequencespresent in one or more naturally occurring cadherins, and cyclicpeptides having specificity may be identified using the representativescreens provided herein. For example, it has been found, within thecontext of the present invention, that cyclic peptides that containadditional residues derived from the native N-cadherin sequence disruptN-cadherin mediated interactions with a high degree of specificity(i.e., such peptides do not significantly disrupt E-cadherin mediatedinteractions).

To facilitate the preparation of cyclic peptides having a desiredspecificity, nuclear magnetic resonance (NMR) and computationaltechniques may be used to determine the conformation of a peptide thatconfers a known specificity. NMR is widely used for structural analysisof both peptidyl and non-peptidyl compounds. Nuclear OverhauserEnhancements (NOE's), coupling constants and chemical shifts depend onthe conformation of a compound. NOE data provide the distance betweenprotons through space and across the ring of the cyclic peptide, and canbe used to calculate the lowest energy conformation for the CARsequence. Cyclic peptides are conformationally restricted and exist inthe active conformation a much higher percentage of the time than to thecorresponding linear peptides. Linear peptides in solution exist in manyconformations. Using a cyclic peptide, it is possible to fix the peptidein the active conformation. Conformation may then be correlated withtissue specificity to permit the identification of peptides that aresimilarly tissue specific or have enhanced tissue specificity.

As noted above, multiple CAR sequences may be present within amodulating agent. In some cases, multiple HAV sequences may be present,preferably separated by a linker as described below. Other CAR sequencesmay also, or alternatively, be included within a modulating agent. Ingeneral, a modulating agent may comprise any sequence specifically boundby an adhesion molecule. As used herein, an “adhesion molecule” is anymolecule that mediates cell adhesion via a receptor on the cell'ssurface. Adhesion molecules include members of the cadherin genesuperfamily that are not classical cadherins (e.g., proteins that do notcontain an HAV sequence and/or one or more of the other characteristicsrecited above for classical cadherins, such as OB-cadherin), as well asintegrins and members of the immunoglobulin supergene family, such asN-CAM Preferred CAR sequences for inclusion within a modulating agentinclude Arg-Gly-Asp (RGD), which is bound by integrins (see Cardarelliet al., J. Biol. Chem. 267:23159-64, 1992); Tyr-Ile-Gly-Ser-Arg (YIGSR;SEQ ID NO:21), which is bound by α6β1 integrin; KYSFNYDGSE (SEQ IDNO:22), which is bound by N-CAM; the N-CAM heparin sulfate-binding siteIWKHKGRDVILKKDVRF (SEQ ID NO:23). Another preferred CAR sequence is theOB-cadherin CAR sequence DDK. A variety of peptides comprising thissequence may be included, such as IDDK (SEQ ID NO:24), DDKS (SEQ IDNO:25), VIDDK (SEQ ID NO:26), IDDKS (SEQ ID NO:27), VIDDKS (SEQ IDNO:28), DDKSG (SEQ ID NO:29), IDDKSG (SEQ ID NO:30), VIDDKSG (SEQ IDNO:31), FVIDDK (SEQ ID NO:32), FVIDDKS (SEQ ID NO:33), FVIDDKSG (SEQ IDNO:34), IFVIDDK (SEQ ID NO:35), IFVIDDKS (SEQ ID NO:36), or IFVIDDKSG(SEQ ID NO:37).

Linkers may, but need not, be used to separate CAR sequences and/orantibody sequences within a modulating agent. Linkers may also, oralternatively, be used to attach one or more modulating agents to asupport molecule or material, as described below. A linker may be anymolecule (including peptide and/or non-peptide sequences as well assingle amino acids or other molecules), that does not contain a CARsequence and that can be covalently linked to at least two peptidesequences. Using a linker, HAV-containing cyclic peptides and otherpeptide or protein sequences may be joined head-to-tail (i.e., thelinker may be covalently attached to the carboxyl or amino group of eachpeptide sequence), head-to-side chain and/or tail-to-side chain.Modulating agents comprising one or more linkers may form linear orbranched structures. Within one embodiment, modulating agents having abranched structure comprise three different CAR sequences, such as RGD,YIGSR (SEQ ID NO:21) and HAV, one or more of which are present within acyclic peptide. Within another embodiment, modulating agents having abranched structure comprise RGD, YIGSR (SEQ ID NO:21), HAV andKYSFNYDGSE (SEQ ID NO:22). Bi-functional modulating agents that comprisean HAV sequence with flanking E-cadherin-specific sequences joined via alinker to an HAV sequence with flanking N-cadherin-specific sequencesare also preferred for certain embodiments.

Linkers preferably produce a distance between CAR sequences between 0.1to 10,000 nm, more preferably about 0.1-400 nm. A separation distancebetween recognition sites may generally be determined according to thedesired function of the modulating agent. For inhibitors of neuriteoutgrowth, the linker distance should be small (0.1-400 nm). Forenhancers of neurite outgrowth, the linker distance should be 400-10,000nm. One linker that can be used for such purposes is(H₂N(CH₂)_(n)CO₂H)_(m), or derivatives thereof, where n ranges from 1 to10 and m ranges from 1 to 4000. For example, if glycine (H₂NCH₂CO₂H) ora multimer thereof is used as a linker, each glycine unit corresponds toa linking distance of 2.45 angstroms, or 0.245 nm, as determined bycalculation of its lowest energy conformation when linked to other aminoacids using molecular modeling techniques. Similarly, aminopropanoicacid corresponds to a linking distance of 3.73 angstroms, aminobutanoicacid to 4.96 angstroms, aminopentanoic acid to 6.30 angstroms and aminohexanoic acid to 6.12 angstroms. Other linkers that may be used will beapparent to those of ordinary skill in the art and include, for example,linkers based on repeat units of 2,3-diaminopropanoic acid, lysineand/or ornithine. 2,3-Diaminopropanoic acid can provide a linkingdistance of either 2.51 or 3.11 angstroms depending on whether theside-chain amino or terminal amino is used in the linkage. Similarly,lysine can provide linking distances of either 2.44 or 6.95 angstromsand ornithine 2.44 or 5.61 angstroms. Peptide and non-peptide linkersmay generally be incorporated into a modulating agent using anyappropriate method known in the art.

Modulating agents that inhibit neurite outgrowth typically contain oneHAV sequence or multiple HAV sequences, which may be adjacent to oneanother (i.e., without intervening sequences) or in close proximity(i.e., separated by peptide and/or non-peptide linkers to give adistance between the CAR sequences that ranges from about 0.1 to 400nm). Within one such embodiment, the cyclic peptide contains two HAVsequences. Such a modulating agent may additionally comprise a CARsequence for one or more different adhesion molecules (including, butnot limited to, other CAMs) and/or one or more antibodies or fragmentsthereof that bind to such sequences. Linkers may, but need not, be usedto separate such CAR sequence(s) and/or antibody sequence(s) from theHAV sequence(s) and/or each other. Such modulating agents may generallybe used within methods in which it is desirable to simultaneouslydisrupt cell adhesion mediated by multiple adhesion molecules. Withincertain preferred embodiments, the second CAR sequence is derived fromfibronectin and is recognized by an integrin (i.e., RGD; see Cardarelliet al., J. Biol. Chem. 267:23159-23164, 1992). Other preferred CARsequences include YIGSR (SEQ ID NO:21) and KYSFNYDGSE (SEQ ID NO:22).One or more antibodies, or fragments thereof, may similarly be usedwithin such embodiments.

Modulating agents that enhance cell adhesion may contain multiple HAVsequences, and/or antibodies that specifically bind to such sequences,joined by linkers as described above. Enhancement of cell adhesion mayalso be achieved by attachment of multiple modulating agents to asupport molecule or material, as discussed further below. Suchmodulating agents may additionally comprise one or more CAR sequence forone or more different adhesion molecules (including, but not limited to,other CAMs) and/or one or more antibodies or fragments thereof that bindto such sequences, to enhance cell adhesion mediated by multipleadhesion molecules.

Modulating agents and cyclic peptides as described herein may compriseresidues of L-amino acids, D-amino acids, or any combination thereof.Amino acids may be from natural or non-natural sources, provided that atleast one amino group and at least one carboxyl group are present in themolecule; α- and β-amino acids are generally preferred. The 20 L-aminoacids commonly found in proteins are identified herein by theconventional three-letter or one-letter abbreviations indicated in Table1, and the corresponding D-amino acids are designated by a lower caseone letter symbol. Modulating agents and cyclic peptides may alsocontain one or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonylation), with or without any of a wide variety ofside-chain modifications and/or substitutions (e.g., methylation,benzylation, t-butylation, tosylation, alkoxycarbonylation, and thelike). Preferred derivatives include amino acids having an N-acetylgroup (such that the amino group that represents the N-terminus of thelinear peptide prior to cyclization is acetylated) and/or a C-terminalamide group (i.e., the carboxy terminus of the linear peptide prior tocyclization is amidated). Residues other than common amino acids thatmay be present with a cyclic peptide include, but are not limited to,penicillamine, β,β-tetramethylene cysteine, β,β-pentamethylene cysteine,β-mercaptopropionic acid, β,β-pentamethylene-β-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, α-aminoadipic acid, m-aminomethylbenzoic acid and(α,β-diaminopropionic acid.

TABLE 1 Amino acid one-letter and three-letter abbreviations A AlaAlanine R Arg Arginine D Asp Aspartic acid N Asn Asparagine C CysCysteine Q Gln Glutamine E Glu Glutamic acid G Gly Glycine H HisHistidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met Methionine FPhe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W TrpTryptophan Y Tyr Tyrosine V Val Valine

Modulating agents and cyclic peptides as described herein may besynthesized by methods well known in the art, including recombinant DNAmethods and chemical synthesis. Chemical synthesis may generally beperformed using standard solution phase or solid phase peptide synthesistechniques, in which a peptide linkage occurs through the directcondensation of the α-amino group of one amino acid with the α-carboxygroup of the other amino acid with the elimination of a water molecule.Peptide bond synthesis by direct condensation, as formulated above,requires suppression of the reactive character of the amino group of thefirst and of the carboxyl group of the second amino acid. The maskingsubstituents must permit their ready removal, without inducing breakdownof the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (see Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy.The Boc strategy uses a 1% cross-linked polystyrene resin. The standardprotecting group for α-amino functions is the tert-butyloxycarbonyl(Boc) group. This group can be removed with dilute solutions of strongacids such as 25% trifluoroacetic acid (TFA). The next Boc-amino acid istypically coupled to the amino acyl resin using dicyclohexylcarbodiimide(DCC). Following completion of the assembly, the peptide-resin istreated with anhydrous HF to cleave the benzyl ester link and liberatethe free peptide. Side-chain functional groups are usually blockedduring synthesis by benzyl-derived blocking groups, which are alsocleaved by HF. The free peptide is then extracted from the resin with asuitable solvent, purified and characterized. Newly synthesized peptidescan be purified, for example, by gel filtration, HPLC, partitionchromatography and/or ion-exchange chromatography, and may becharacterized by, for example, mass spectrometry or amino acid sequenceanalysis. In the Boc strategy, C-terminal amidated peptides can beobtained using benzhydrylamine or methylbenzhydrylamine resins, whichyield peptide amides directly upon cleavage with HF.

In the procedures discussed above, the selectivity of the side-chainblocking groups and of the peptide-resin link depends upon thedifferences in the rate of acidolytic cleavage. Orthoganol systems havebeen introduced in which the side-chain blocking groups and thepeptide-resin link are completely stable to the reagent used to removethe α-protecting group at each step of the synthesis. The most common ofthese methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach.Within this method, the side-chain protecting groups and thepeptide-resin link are completely stable to the secondary amines usedfor cleaving the N-α-Fmoc group. The side-chain protection and thepeptide-resin link are cleaved by mild acidolysis. The repeated contactwith base makes the Merrifield resin unsuitable for Fmoc chemistry, andp-alkoxybenzyl esters linked to the resin are generally used.Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Following synthesis of a linear peptide, with or without N-acetylationand/or C-amidation, cyclization may be achieved by any of a variety oftechniques well known in the art. Within one embodiment, a bond may begenerated between reactive amino acid side chains. For example, adisulfide bridge may be formed from a linear peptide comprising twothiol-containing residues by oxidizing the peptide using any of avariety of methods. Within one such method, air oxidation of thiols cangenerate disulfide linkages over a period of several days using eitherbasic or neutral aqueous media. The peptide is used in high dilution tominimize aggregation and intermolecular side reactions. This methodsuffers from the disadvantage of being slow but has the advantage ofonly producing H₂O as a side product. Alternatively, strong oxidizingagents such as I₂ and K₃Fe(CN)₆ can be used to form disulfide linkages.Those of ordinary skill in the art will recognize that care must betaken not to oxidize the sensitive side chains of Met, Tyr, Trp or His.Cyclic peptides produced by this method require purification usingstandard techniques, but this oxidation is applicable at acid pHs. Byway of example, strong oxidizing agents can be used to perform thecyclization shown below (SEQ ID NO: 38), in which the underlined portionis cyclized:

FmocCysAsp(t-Bu)GlyTyr(t-Bu)ProLys(Boc)Asp(t-Bu)CysLys(t-Bu)Gly-OMe

→

FmocCysAsp(t-Bu)GlyTyr(t-Bu)ProLys(Boc)Asp(t-Bu)CysLys(t-Bu)Gly-OMe

Oxidizing agents also allow concurrent deprotection/oxidation ofsuitable S-protected linear precursors to avoid premature, nonspecificoxidation of free cysteine, as shown below (SEQ ID NO:39), where X andY=S-Trt or S-Acm:

BocCys(X)GlyAsnLeuSer(t-Bu)Thr(t-Bu)Cys(Y)MetLeuGlyOH→

BocCysGlyAsnLeuSer(t-Bu)Thr(t-Bu)CysMetLeuGlyOH

DMSO, unlike I₂ and K₃Fe(CN)₆, is a mild oxidizing agent which does notcause oxidative side reactions of the nucleophilic amino acids mentionedabove. DMSO is miscible with H₂O at all concentrations, and oxidationscan be performed at acidic to neutral pHs with harmless byproducts.Methyltrichlorosilane-diphenylsulfoxide may alternatively be used as anoxidizing agent, for concurrent deprotection/oxidation of S-Acm, S-Tacmor S-t-Bu of cysteine without affecting other nucleophilic amino acids.There are no polymeric products resulting from intermolecular disulfidebond formation. In the example below (SEQ ID NO:40), X is Acm, Tacm ort-Bu:

H-Cys(X)TyrIleGlnAsnCys(X)ProLeuGly-NH₂→

H-CysTyrIleGlnAsnCysProLeuGly-NH₂

Suitable thiol-containing residues for use in such oxidation methodsinclude, but are not limited to, cysteine, β,β-dimethyl cysteine(penicillamine or Pen), β,β-tetramethylene cysteine (Tmc),β,β-pentamethylene cysteine (Pmc), β-mercaptopropionic acid (Mpr),β,β-pentamethylene-β-mercaptopropionic acid (Pmp), 2-mercaptobenzene,2-mercaptoaniline and 2-mercaptoproline. Peptides containing suchresidues are illustrated by the following representative formulas, inwhich the underlined portion is cyclized, N-acetyl groups are indicatedby N-Ac and C-terminal amide groups are represented by —NH₂:

i) N-Ac-Cys-His-Ala-Val-Cys-NH₂ (SEQ ID NO:10)

ii) N-Ac-Cys-Ala-His-Ala-Val-Asp-Ile-Cys-NH₂ (SEQ ID NO:14)

iii) N-Ac-Cys-Ser-His-Ala-Val-Cys-NH₂ (SEQ ID NO:41)

iv) N-Ac-Cys-His-Ala-Val-Ser-Cys-NH₂ (SEQ ID NO:42)

v) N-Ac-Cys-Ala-His-Ala-Val-Asp-Cys-NH₂ (SEQ ID NO:13)

vi) N-Ac-Cys-Ser-His-Ala-Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:43)

vii) N-Ac-Cys-His-Ala-Val-Ser-Cys-OH (SEQ ID NO:42)

viii) H-Cys-Ala-His-Ala-Val-Asp-Cys-NH₂ (SEQ ID NO:13)

ix) N-Ac-Cys-His-Ala-Val-Pen-NH₂ (SEQ ID NO:44)

x) N-Ac-Ile-Tmc-Tyr-Ser-His-Ala-Val-Ser-Cys-Glu-NH₂ (SEQ ID NO:45)

xi) N-Ac-Ile-Pmc-Tyr-Ser-His-Ala-Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:46)

xii) Mpr-Tyr-Ser-His-Ala- Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:47)

xiii) Pmp-Tyr-Ser-His-Ala-Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:48)

xii)

xiii)

It will be readily apparent to those of ordinary skill in the art that,within each of these representative formulas, any of the abovethiol-containing residues may be employed in place of one or both of thethiol-containing residues recited.

Within another embodiment, cyclization may be achieved by amide bondformation. For example, a peptide bond may be formed between terminalfunctional groups (i.e., the amino and carboxy termini of a linearpeptide prior to cyclization). Two such cyclic peptides are AHAVDI (SEQID NO:19) and SHAVSS (SEQ ID NO:49), with or without an N-terminalacetyl group and/or a C-terminal amide. Within another such embodiment,the linear peptide comprises a D-amino acid (e.g., HAVsS; SEQ ID NO:50).Alternatively, cyclization may be accomplished by linking one terminusand a residue side chain or using two side chains, with or without anN-terminal acetyl group and/or a C-terminal amide. Residues capable offorming a lactam bond include lysine, ornithine (Orn), α-amino adipicacid, m-aminomethylbenzoic acid, α,β-diaminopropionic acid, glutamate oraspartate.

Methods for forming amide bonds are well known in the art and are basedon well established principles of chemical reactivity. Within one suchmethod, carbodiimide-mediated lactam formation can be accomplished byreaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resultingin the formation of an O-acylurea that can be reacted immediately withthe free amino group to complete the cyclization. The formation of theinactive N-acylurea, resulting from O→N migration, can be circumventedby converting the O-acylurea to an active ester by reaction with anN-hydroxy compound such as 1-hydroxybenzotriazole, 1-hydroxysuccinimide,1-hydroxynorbornene carboxamide or ethyl 2-hydroximino-2-cyanoacetate.In addition to minimizing O→N migration, these additives also serve ascatalysts during cyclization and assist in lowering racemization.Alternatively, cyclization can be performed using the azide method, inwhich a reactive azide intermediate is generated from an alkyl ester viaa hydrazide. Hydrazinolysis of the terminal ester necessitates the useof a t-butyl group for the protection of side chain carboxyl functionsin the acylating component. This limitation can be overcome by usingdiphenylphosphoryl acid (DPPA), which furnishes an azide directly uponreaction with a carboxyl group. The slow reactivity of azides and theformation of isocyanates by their disproportionation restrict theusefulness of this method. The mixed anhydride method of lactamformation is widely used because of the facile removal of reactionby-products. The anhydride is formed upon reaction of the carboxylateanion with an alkyl chloroformate or pivaloyl chloride. The attack ofthe amino component is then guided to the carbonyl carbon of theacylating component by the electron donating effect of the alkoxy groupor by the steric bulk of the pivaloyl chloride t-butyl group, whichobstructs attack on the wrong carbonyl group. Mixed anhydrides withphosphoric acid derivatives have also been successfully used.Alternatively, cyclization can be accomplished using activated esters.The presence of electron withdrawing substituents on the alkoxy carbonof esters increases their susceptibility to aminolysis. The highreactivity of esters of p-nitrophenol, N-hydroxy compounds andpolyhalogenated phenols has made these “lactive esters” useful in thesynthesis of amide bonds. The last few years have witnessed thedevelopment of benzotriazolyloxytris-(dimethylamino)phosphoniumhexafluorophosphonate (BOP) and its congeners as advantageous couplingreagents. Their performance is generally superior to that of the wellestablished carbodiimide amide bond formation reactions.

Within a further embodiment, a thioether linkage may be formed betweenthe side chain of a thiol-containing residue and an appropriatelyderivatized α-amino acid. By way of example, a lysine side chain can becoupled to bromoacetic acid through the carbodiimide coupling method(DCC, EDAC) and then reacted with the side chain of any of the thiolcontaining residues mentioned above to form a thioether linkage. Inorder to form dithioethers, any two thiol containing side-chains can bereacted with dibromoethane and diisopropylamine in DMF. Examples ofthiol-containing linkages are shown below:

Cyclization may also be achieved using δ₁,δ₁,-Ditryptophan (i.e.,Ac-Trp-Gly-Gly-Trp-OMe) (SEQ ID NO:51), as shown below:

Representative structures of cyclic peptides are provided in FIG. 3.Within FIG. 3, certain cyclic peptides having the ability to modulatecell adhesion (shown on the left) are paired with similar inactivestructures (on the right). The structures and formulas recited hereinare provided solely for the purpose of illustration, and are notintended to limit the scope of the cyclic peptides described herein.

As noted above, a modulating agent may consist entirely of one or morecyclic peptides, or may contain additional peptide and/or non-peptidesequences, which may be linked to the cyclic peptide(s) usingconventional techniques. Peptide portions may be synthesized asdescribed above or may be prepared using recombinant methods. Withinsuch methods, all or part of a modulating agent can be synthesized inliving cells, using any of a variety of expression vectors known tothose of ordinary skill in the art to be appropriate for the particularhost cell. Suitable host cells may include bacteria, yeast cells,mammalian cells, insect cells, plant cells, algae and other animal cells(e.g., hybridoma, CHO, myeloma). The DNA sequences expressed in thismanner may encode portions of an endogenous cadherin or other adhesionmolecule. Such sequences may be prepared based on known cDNA or genomicsequences (see Blaschuk et al., J. Mol. Biol. 211:679-682, 1990), orfrom sequences isolated by screening an appropriate library with probesdesigned based on the sequences of known cadherins. Such screens maygenerally be performed as described in Sambrook et al., MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y., 1989 (and references cited therein). Polymerasechain reaction (PCR) may also be employed, using oligonucleotide primersin methods well known in the art, to isolate nucleic acid moleculesencoding all or a portion of an endogenous adhesion molecule. Togenerate a nucleic acid molecule encoding a peptide portion of amodulating agent, an endogenous sequence may be modified using wellknown techniques. For example, portions encoding one or more CARsequences may be joined, with or without separation by nucleic acidregions encoding linkers, as discussed above. Alternatively, portions ofthe desired nucleic acid sequences may be synthesized using well knowntechniques, and then ligated together to form a sequence encoding aportion of the modulating agent.

As noted above, portions of a modulating agent may comprise an antibody,or antigen-binding fragment thereof, that specifically binds to a CARsequence. As used herein, an antibody, or antigen-binding fragmentthereof, is said to “specifically bind” to a CAR sequence (with orwithout flanking amino acids) if it reacts at a detectable level(within, for example, an ELISA, as described by Newton et al., Develop.Dynamics 197:1-13, 1993) with a peptide containing that sequence, anddoes not react detectably with peptides containing a different CARsequence or a sequence in which the order of amino acid residues in thecadherin CAR sequence and/or flanking sequence is altered.

Antibodies and fragments thereof may be prepared using standardtechniques. See, e.g. Harlow and Lane, Antibodies. A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogencomprising a CAR sequence is initially injected into any of a widevariety of mammals (e.g, mice, rats, rabbits, sheep or goats). Smallimmunogens (i.e., less than about 20 amino acids) should be joined to acarrier protein, such as bovine serum albumin or keyhole limpethemocyanin. Following one or more injections, the animals are bledperiodically. Polyclonal antibodies specific for the CAR sequence maythen be purified from such antisera by, for example, affinitychromatography using the modulating agent or antigenic portion thereofcoupled to a suitable solid support.

Monoclonal antibodies specific for the cyclic peptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, thesemethods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity from spleen cellsobtained from an animal immunized as described above. The spleen cellsare immortalized by, for example, fusion with a myeloma cell fusionpartner, preferably one that is syngeneic with the immunized animal.Single colonies are selected and their culture supernatants tested forbinding activity against the polypeptide. Hybridomas having highreactivity and specificity are preferred. Monoclonal antibodies may bespecific for particular cadherins (e.g., antibodies may bind toN-cadherin, without binding significantly to E-cadherin).

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies, with or without the use of various techniques knownin the art to enhance the yield. Contaminants may be removed from theantibodies by conventional techniques, such as chromatography, gelfiltration, precipitation, and extraction. Antibodies having the desiredactivity may generally be identified using immunofluorescence analysesof tissue sections, cell or other samples where the target cadherin islocalized.

Within certain embodiments, monoclonal antibodies may be specific forparticular cadherins (e.g., the antibodies bind to N-cadherin, but donot bind significantly to E-cadherin). Such antibodies may be preparedas described above, using an immunogen that comprises (in addition tothe HAV sequence) sufficient flanking sequence to generate the desiredspecificity (e.g., 5 amino acids on each side is generally sufficient).One representative immunogen is the 15-mer FHLRAHAVDINGNQV-NH₂ (SEQ IDNO:52), linked to KLH (see Newton et al., Dev. Dynamics 197:1-13, 1993).To evaluate the specificity of a particular antibody, representativeassays as described herein and/or conventional antigen-binding assaysmay be employed. Such antibodies may generally be used for therapeutic,diagnostic and assay purposes, as described herein. For example, suchantibodies may be linked to a drug and administered to a mammal totarget the drug to a particular cadherin-expressing cell, such as aleukemic cell in the blood.

The use of antigen-binding fragments of antibodies may be preferredwithin certain embodiments. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies. A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; see especially page 309) and digested bypapain to yield Fab and Fc fragments. The Fab and Fc fragments may beseparated by affinity chromatography on protein A bead columns (Harlowand Lane, 1988, pages 628-29).

EVALUATION OF MODULATING AGENT ACTIVITY

As noted above, cyclic peptides and other modulating agents as describedherein are capable of modulating (i.e., enhancing, inhibiting andlordirecting) cadherin-mediated neurite outgrowth. Within a representativeneurite outgrowth assay, neurons may be cultured on a monolayer of cells(e.g., 3T3) that express N-cadherin. Neurons grown on such cells (undersuitable conditions and for a sufficient period of time) extend longerneurites than neurons cultured on cells that do not express N-cadherin.For example, neurons may be cultured on monolayers of 3T3 cellstransfected with CDNA encoding N-cadherin essentially as described byDoherty and Walsh, Curr. Op. Neurobiol. 4:49-55, 1994; Williams et al.,Neuron 13:583-594, 1994; Hall et al., Cell Adhesion and Commun.3:441-450, 1996; Doherty and Walsh, Mol. Cell. Neurosci. 8:99-111, 1994;and Safell et al., Neuron 18:231-242, 1997. Briefly, monolayers ofcontrol 3T3 fibroblasts and 3T3 fibroblasts that express N-cadherin maybe established by overnight culture of 80,000 cells in individual wellsof an 8-chamber well tissue culture slide. 3000 cerebellar neuronsisolated from post-natal day 3 mouse brains may be cultured for 18 hourson the various monolayers in control media (SATO/2%FCS), or mediasupplemented with various concentrations of the modulating agent orcontrol peptide. The cultures may then be fixed and stained for GAP43which specifically binds to the neurons and their neurites. The lengthof the longest neurite on each GAP43 positive neuron may be measured bycomputer assisted morphometry.

A modulating agent that modulates N-cadherin-mediated cell adhesion mayinhibit or enhance such neurite outgrowth. Under the conditionsdescribed above, the presence of 500 μg/mL of a modulating agent thatdisrupts neural cell adhesion should result in a decrease in the meanneurite length by at least 50%, relative to the length in the absence ofmodulating agent or in the presence of a negative control peptide.Alternatively, the presence of 500 μg/mL of a modulating agent thatenhances neural cell adhesion should result in an increase in the meanneurite length by at least 50%.

MODULATING AGENT MODIFICATION AND FORMULATIONS

A modulating agent as described herein may, but need not, be linked toone or more additional molecules. In particular, as discussed below, itmay be beneficial for certain applications to link multiple modulatingagents (which may, but need not, be identical) to a support molecule(e.g., keyhole limpet hemocyanin) or a solid support, such as apolymeric matrix (which may be formulated as a membrane ormicrostructure, such as an ultra thin film), a container surface (e.g.,the surface of a tissue culture plate or the interior surface of abioreactor), or a bead or other particle, which may be prepared from avariety of materials including glass, plastic or ceramics. For certainapplications, biodegradable support materials are preferred, such ascellulose and derivatives thereof, collagen, spider silk or any of avariety of polyesters (e.g., those derived from hydroxy acids and/orlactones) or sutures (see U.S. Pat. No. 5,245,012). Within certainembodiments, modulating agents and molecules comprising other CARsequence(s) (e.g., an RGD sequence) may be attached to a support such asa polymeric matrix, preferably in an alternating pattern.

Suitable methods for linking a modulating agent to a support materialwill depend upon the composition of the support and the intended use,and will be readily apparent to those of ordinary skill in the art.Attachment may generally be achieved through noncovalent association,such as adsorption or affinity or, preferably, via covalent attachment(which may be a direct linkage between a modulating agent and functionalgroups on the support, or may be a linkage by way of a cross-linkingagent or linker). Attachment of a modulating agent by adsorption may beachieved by contact, in a suitable buffer, with a solid support for asuitable amount of time. The contact time varies with temperature, butis generally between about 5 seconds and 1 day, and typically betweenabout 10 seconds and 1 hour.

Covalent attachment of a modulating agent to a molecule or solid supportmay generally be achieved by first reacting the support material with abifunctional reagent that will also react with a functional group, suchas a hydroxyl, thiol, carboxyl, ketone or amino group, on the modulatingagent. For example, a modulating agent may be bound to an appropriatepolymeric support or coating using benzoquinone, by condensation of analdehyde group on the support with an amine and an active hydrogen onthe modulating agent or by condensation of an amino group on the supportwith a carboxylic acid on the modulating agent. A preferred method ofgenerating a linkage is via amino groups using glutaraldehyde. Amodulating agent may be linked to cellulose via ester linkages.Similarly, amide linkages may be suitable for linkage to other moleculessuch as keyhole limpet hemocyanin or other support materials. Multiplemodulating agents and/or molecules comprising other CAR sequences may beattached, for example, by random coupling, in which equimolar amounts ofsuch molecules are mixed with a matrix support and allowed to couple atrandom.

Although modulating agents as described herein may preferentially bindto specific tissues or cells, and thus may be sufficient to target adesired site in vivo, it may be beneficial for certain applications toinclude an additional targeting agent. Accordingly, a targeting agentmay also, or alternatively, be linked to a modulating agent tofacilitate targeting to one or more specific tissues. As used herein, a“targeting agent,” may be any substance (such as a compound or cell)that, when linked to a modulating agent enhances the transport of themodulating agent to a target tissue, thereby increasing the localconcentration of the modulating agent. Targeting agents includeantibodies or fragments thereof, receptors, ligands and other moleculesthat bind to cells of, or in the vicinity of, the target tissue. Knowntargeting agents include serum hormones, antibodies against cell surfaceantigens, lectins, adhesion molecules, tumor cell surface bindingligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes andthose drugs and proteins that bind to a desired target site. An antibodytargeting agent may be an intact (whole) molecule, a fragment thereof,or a functional equivalent thereof. Examples of antibody fragments areF(ab′)2, -Fab′, Fab and F[v] fragments, which may be produced byconventional methods or by genetic or protein engineering. Linkage isgenerally covalent and may be achieved by, for example, directcondensation or other reactions, or by way of bi- or multi-functionallinkers. Within other embodiments, it may also be possible to target apolynucleotide encoding a modulating agent to a target tissue, therebyincreasing the local concentration of modulating agent. Such targetingmay be achieved using well known techniques, including retroviral andadenoviral infection.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a modulating agent. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds.

Within certain aspects of the present invention, one or more modulatingagents as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or moremodulating agents in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. A modulating agent (alone or incombination with a targeting agent and/or drug) may, but need not, beencapsulated within liposomes using well known technology. Compositionsof the present invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration. For certain topical applications,formulation as a cream or lotion, using well known components, ispreferred.

For certain embodiments, as discussed below, a pharmaceuticalcomposition may further comprise a modulator of cell adhesion that ismediated by one or more molecules other than cadherins. Such modulatorsmay generally be prepared as described above, incorporating one or morenon-cadherin CAR sequences and/or antibodies thereto in place of thecadherin CAR sequences and antibodies. Such compositions areparticularly useful for situations in which it is desirable to inhibitcell adhesion mediated by multiple cell-adhesion molecules, such asother members of the cadherin gene superfamily that are not classicalcadherins (e.g., OB-cadherin); integrins; members of the immunoglobulinsupergene family, such as N-CAM; and other uncategorized transmembraneproteins. Preferred CAR sequences for use within such a modulatorinclude RGD, YIGSR (SEQ ID NO:21), KYSFNYDGSE (SEQ ID NO:22),IWKHKGRDVILKKDVRF (SEQ ID NO:23), and the OB-cadherin CAR sequence DDK.As noted above, a variety of peptides comprising the OB-cadherin CARsequence may be included, such as IDDK (SEQ ID NO:24), DDKS (SEQ IDNO:25), VIDDK (SEQ ID NO:26), IDDKS (SEQ ID NO:27), VIDDKS (SEQ IDNO:28), DDKSG (SEQ ID NO:29), IDDKSG (SEQ ID NO:30), VIDDKSG (SEQ IDNO:31), FVIDDK (SEQ ID NO:32), FVIDDKS (SEQ ID NO:33), FVIDDKSG (SEQ IDNO:34), IFVIDDK (SEQ ID NO:35), IFVIDDKS (SEQ ID NO:36), or IFVIDDKSG(SEQ ID NO:37).

A pharmaceutical composition may also contain one or more drugs, whichmay be linked to a modulating agent or may be free within thecomposition. Virtually any drug may be administered in combination witha cyclic peptide as described herein, for a variety of purposes asdescribed below. Examples of types of drugs that may be administeredwith a cyclic peptide include analgesics, anesthetics, antianginals,antifungals, antibiotics, anticancer drugs (e.g., taxol or mitomycin C),antiinflammatories (e.g., ibuprofen and indomethacin), anthelmintics,antidepressants, antidotes, antiemetics, antihistamines,antihypertensives, antimalarials, antimicrotubule agents (e.g.,colchicine or vinca alkaloids), antimigraine agents, antimicrobials,antiphsychotics, antipyretics, antiseptics, anti-signaling agents (e.g.,protein kinase C inhibitors or inhibitors of intracellular calciummobilization), antiarthritics, antithrombin agents, antituberculotics,antitussives, antivirals, appetite suppressants, cardioactive drugs,chemical dependency drugs, cathartics, chemotherapeutic agents,coronary, cerebral or peripheral vasodilators, contraceptive agents,depressants, diuretics, expectorants, growth factors, hormonal agents,hypnotics, immunosuppression agents, narcotic antagonists,parasympathomimetics, sedatives, stimulants, sympathomimetics, toxins(e.g., cholera toxin), tranquilizers and urinary anti infectives.

For imaging purposes, any of a variety of diagnostic agents may beincorporated into a pharmaceutical composition, either linked to amodulating agent or free within the composition. Diagnostic agentsinclude any substance administered to illuminate a physiologicalfunction within a patient, while leaving other physiological functionsgenerally unaffected. Diagnostic agents include metals, radioactiveisotopes and radioopaque agents (e.g., gallium, technetium, indium,strontium, iodine, barium, bromine and phosphorus-containing compounds),radiolucent agents, contrast agents, dyes (e.g., fluorescent dyes andchromophores) and enzymes that catalyze a colorimetric or fluorometricreaction. In general, such agents may be attached using a variety oftechniques as described above, and may be present in any orientation.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of cyclic peptide followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a cyclic peptidedispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane (see, e.g., European PatentApplication 710,491 A). Carriers for use within such formulations arebiocompatible, and may also be biodegradable; preferably the formulationprovides a relatively constant level of cyclic peptide release. Theamount of cyclic peptide contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the condition to be treated (or prevented).Appropriate dosages and the duration and frequency of administrationwill be determined by such factors as the condition of the patient, thetype and severity of the patient's disease and the method ofadministration. In general, an appropriate dosage and treatment regimenprovides the modulating agent(s) in an amount sufficient to providetherapeutic and/or prophylactic benefit. Within particularly preferredembodiments of the invention, a modulating agent or pharmaceuticalcomposition as described herein may be administered at a dosage rangingfrom 0.001 to 50 mg/kg body weight, preferably from 0.1 to 20 mg/kg, ona regimen of single or multiple daily doses. For topical administration,a cream typically comprises an amount of modulating agent ranging from0.00001% to 1%, preferably 0.0001% to 0.2%, and more preferably from0.0001% to 0.002%. Fluid compositions typically contain about 10 ng/mlto 5 mg/ml, preferably from about 10 μg to 2 mg/mL cyclic peptide.Appropriate dosages may generally be determined using experimentalmodels and/or clinical trials. In general, the use of the minimum dosagethat is sufficient to provide effective therapy is preferred. Patientsmay generally be monitored for therapeutic effectiveness using assayssuitable for the condition being treated or prevented, which will befamiliar to those of ordinary skill in the art.

MODULATING AGENT METHODS OF USE

In general, the modulating agents and compositions described herein maybe used for modulating neurite outgrowth of cadherin-expressing neuralcells in vitro and/or in vivo. As noted above, modulating agents forpurposes that involve the disruption of cadherin-mediated neuriteoutgrowth may comprise a cyclic peptide containing a single HAVsequence, multiple HAV sequences in close proximity and/or an antibody(or an antigen-binding fragment thereof) that recognizes a cadherin CARsequence. When it is desirable to also disrupt cell adhesion mediated byother adhesion molecules, a modulating agent may additionally compriseone or more CAR sequences bound by such adhesion molecules (and/orantibodies or fragments thereof that bind such sequences), preferablyseparated from each other and from the HAV sequence by linkers. As notedabove, such linkers may or may not comprise one or more amino acids. Forenhancing neurite outgrowth, a modulating agent may contain multiple HAVsequences or antibodies (or fragments), preferably separated by linkers,and/or may be linked to a single molecule or to a support material asdescribed above. Within the methods described herein, one or moremodulating agents may generally be administered alone, or within apharmaceutical composition. In each specific method described herein, asnoted above, a targeting agent may be employed to increase the localconcentration of modulating agent at the target site.

Modulating agents may generally be used, within certain aspects, toenhance and/or direct neurological growth. In one aspect, neuriteoutgrowth may be enhanced and/or directed by contacting a neuron withone or more modulating agents. Preferred modulating agents for usewithin such methods are linked to a polymeric matrix or other supportand include those peptides without substantial flanking sequences, asdescribed above. In particularly preferred embodiments, the modulatingagent comprises a cyclic peptide such as N-Ac-CHAVC-NH₂ (SEQ ID NO:10),N-Ac-CHAVDC-NH₂ (SEQ ID NO:11), N-Ac-CAHAVC-NH₂ (SEQ ID NO:12),N-Ac-CAHAVDC-NH₂ (SEQ ID NO:13), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:14),N-Ac-CRAHAVDC-NH₂ (SEQ ID NO: 15), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:16),N-Ac-DHAVK-NH₂ (SEQ ID NO:17), N-Ac-KHAVE-NH₂ (SEQ ID NO:18),N-Ac-AHAVDI-NH₂ (SEQ ID NO:20) and derivatives thereof, includingderivatives without the N-acetyl group. In addition, a modulating agentcomprising RGD and/or YIGSR (SEQ ID NO:21), which are bound byintegrins, the cadherin CAR sequence HAV, and/or the N-CAM CAR sequenceKYSFNYDGSE (SEQ ID NO:22) may further facilitate neurite outgrowth.Modulating agents comprising antibodies, or fragments thereof, may beused within this aspect of the present invention without the use oflinkers or support materials. Preferred antibody modulating agentsinclude Fab fragments directed against the N-cadherin CAR sequenceFHLRAHAVDINGNQV-NH₂ (SEQ ID NO:52). Fab fragments directed against theN-CAM CAR sequence KYSFNYDGSE (SEQ ID NO:22) may also be employed,either incorporated into the modulating agent or administeredconcurrently as a separate modulator.

The method of achieving contact and the amount of modulating agent usedwill depend upon the location of the neuron and the extent and nature ofthe outgrowth desired. For example, a neuron may be contacted (e.g., viaimplantation) with modulating agent(s) linked to a support material suchas a suture, fiber nerve guide or other prosthetic device such that theneurite outgrowth is directed along the support material. Alternatively,a tubular nerve guide may be employed, in which the lumen of the nerveguide contains a composition comprising the modulating agent(s). Invivo, such nerve guides or other supported modulating agents may beimplanted using well known techniques to, for example, facilitate thegrowth of severed neuronal connections and/or to treat spinal cordinjuries. It will be apparent to those of ordinary skill in the art thatthe structure and composition of the support should be appropriate forthe particular injury being treated. In vitro, a polymeric matrix maysimilarly be used to direct the growth of neurons onto patternedsurfaces as described, for example, in U.S. Pat. No. 5,510,628.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1

Preparation of Representative Cyclic Peptides

This Example illustrates the solid phase synthesis of representativecyclic peptides.

The peptides were assembled on methylbenzhydrylamine resin (MBHA resin)for the C-terminal amide peptides. The traditional Merrifield resinswere used for any C-terminal acid peptides. Bags of a polypropylene meshmaterial were filled with the resin and soaked in dichloromethane. Theresin packets were washed three times with 5% diisopropylethylamine indichloromethane and then washed with dichloromethane. The packets arethen sorted and placed into a Nalgene bottle containing a solution ofthe amino acid of interest in dichloromethane. An equal amount ofdiisopropylcarbodiimide (DIC) in dichloromethane was added to activatethe coupling reaction. The bottle was shaken for one hour to ensurecompletion of the reaction. The reaction mixture was discarded and thepackets washed with DMF. The N-α-Boc was removed by acidolysis using a55% TFA in dichloromethane for 30 minutes leaving the TFA salt of theα-amino group. The bags were washed and the synthesis completed byrepeating the same procedure while substituting for the correspondingamino acid at the coupling step. Acetylation of the N-terminal wasperformed by reacting the peptide resins with a solution of aceticanhydride in dichloromethane in the presence of diisopropylethylamine.The peptide was then side-chain deprotected and cleaved from the resinat 0° C. with liquid HF in the presence of anisole as a carbocationscavenger.

The crude peptides were purified by reversed-phase high-performanceliquid chromatography. Purified linear precursors of the cyclic peptideswere solubilized in 75% acetic acid at a concentration of 2-10 mg/mL. A10% solution of iodine in methanol was added dropwise until a persistentcoloration was obtained. A 5% ascorbic acid solution in water was thenadded to the mixture until discoloration. The disulfide bridgecontaining compounds were then purified by HPLC and characterized byanalytical HPLC and by mass spectral analysis.

EXAMPLE 2

Disruption of the Ability of Mouse Cerebellar Neurons to Extend Neurites

Three cell adhesion molecules, N-cadherin, N-CAM and L1, are capable ofregulating neurite outgrowth (Doherty and Walsh, Curr. Op. Neurobiol.4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994; Hall et al.,Cell Adhesion and Commun. 3:441-450, 1996; Doherty and Walsh, Mol. Cell.Neurosci. 8:99-111, 1994; Safell et al., Neuron 18:231-242, 1997).Neurons cultured on monolayers of 3T3 cells that have been transfectedwith cDNAs encoding N-cadherin, N-CAM or L1 extend longer neurites thanneurons cultured on 3T3 cells not expressing these cell adhesionmolecules. This Example illustrates the use of a representative cyclicpeptide to inhibit neurite outgrowth.

Neurons were cultured on monolayers of 3T3 cells transfected with cDNAencoding N-cadherin essentially as described by Doherty and Walsh, Curr.Op. Neurobiol. 4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994;Hall et al., Cell Adhesion and Commun. 3:441-450, 1996; Doherty andWalsh, Mol. Cell. Neurosci. 8:99-111, 1994; Safell et al., Neuron18:231-242, 1997. Briefly, monolayers of control 3T3 fibroblasts and 3T3fibroblasts that express N-cadherin were established by overnightculture of 80,000 cells in individual wells of an 8-chamber well tissueculture slide. 3000 cerebellar neurons isolated from post-natal day 3mouse brains were cultured for 18 hours on the various monolayers incontrol media (SATO/2%FCS), or media supplemented with variousconcentrations of the cyclic peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10) or acontrol peptide without the HAV sequence (N-Ac-CHGVC-NH₂; SEQ ID NO:20).The cultures were then fixed and stained for GAP43 which specificallybinds to the neurons and their neurites. The length of the longestneurite on each GAP43 positive neuron was then measured by computerassisted morphometry.

As shown in FIG. 4, culture for 18 hours with N-Ac-CHAVC-NH₂ (SEQ IDNO:10)at a concentration of 500 μg/mL inhibited neurite outgrowth on 3T3cells expressing N-cadherin, whereas the cyclic peptide N-Ac-CHGVC-NH₂(SEQ ID NO:20; also at a concentration of 500 μg/ml) had no effect onthis process. Furthermore, the cyclic peptide N-Ac-CHAVC-NH₂ (SEQ IDNO:10; used at a concentration of 500 μg/ml) did not inhibit neuriteoutgrowth on 3T3 cells not expressing N-cadherin, N-CAM, or L1 (controlcells), thus indicating that the peptide is not toxic and that it has nonon-specific effects on neurite outgrowth (FIG. 4, compare columns 3 and1). These data also indicate that the peptide does not effect integrinfunction.

A dose-response study demonstrated that N-Ac-CHAVC-NH₂ (SEQ ID NO:10)significantly inhibited neurite outgrowth on 3T3 cells expressingN-cadherin at a concentration of 50 μg/mL, and completely inhibitedneurite outgrowth on these cells at a concentration of 500 μg/mL (FIG.5). Finally, N-Ac-CHAVC-NH₂ (SEQ ID NO:10; used at a concentration of500 μg/mL) did not inhibit neurite outgrowth on 3T3 cells expressingeither N-CAM or L1 (FIG. 6). These results indicate that the peptideN-Ac-CHAVC-NH₂ (SEQ ID NO:10) specifically inhibits the function ofN-cadherin. Collectively, the results obtained from these studiesdemonstrate that N-Ac-CHAVC-NH₂ (SEQ ID NO:10) is an effective inhibitorof neurite outgrowth by virtue of its ability to disrupt N-cadherinfunction.

EXAMPLE 3

Disruption of Neurite Outgrowth

This Example further illustrates the use of representative modulatingagents to inhibit neurite outgrowth.

A series of cyclic peptide modulating agents was tested for theirability to inhibit neurite outgrowth. Certain peptides werenon-selective (i.e., not specific for a particular cadherin), whileothers were designed to incorporate flanking sequences of N-cadherin orE-cadherin. The percentage inhibition of neurite outgrowth for eachcompound (at 250 μg/mL) was then evaluated as described in Example 2,except that neurons were isolated from rats, rather than mice.

The results are presented in Table 5 and FIG. 7. The non-selectivepeptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10) significantly inhibited neuriteoutgrowth when the peptide contained N-acetyl and C-amide groups, butnot in the absence of the N-acetyl group. Control peptides comprising anHGV sequence did not inhibit neurite outgrowth detectably. Most of theN-cadherin specific peptides did inhibit neurite outgrowth, but none ofthe E-cadherin specific peptides had a detectable effect. These resultsdemonstrate the specificity of modulating agents comprising cyclicpeptides with flanking sequences in addition to the HAV sequence.

TABLE 5 Evaluation of Peptides for Ability to Inhibit Neurite OutgrowthPeptide Sequence % Inhibition 250 μg/mL Nonselective N-Ac-CHAVC-NH2 (SEQID NO:10) 65% N-Ac-CHGVC-NH2 (SEQ ID NO:20) 0% H-CHAVC-NH2 (SEQ IDNO:10) 0% H-CHGVC-NH2 (SEQ ID NO:20) 0% N-Ac-KHAVD-NH2 (SEQ ID NO:53) 0%N-Ac-KHGVD-NH2 (SEQ ID NO:54) 0% H-KHAVD-NH2 (SEQ ID NO:53) 0%H-KHGVD-NH2 (SEQ ID NO:54) 0% N-cadherin specific N-Ac-CHAVDC-NH2 (SEQID NO:11) 88% N-Ac-CHGVDC-NH2 (SEQ ID NO:55) 0% N-Ac-CAHAVC-NH2 (SEQ IDNO:12) 51% N-Ac-CAHGVC-NH2 (SEQ ID NO:56) 0% N-Ac-CAHAVDIC-NH2 (SEQ IDNO:14) 82% N-Ac-CAHGVDIC-NH2 (SEQ ID NO:57) 0% N-Ac-CRAHAVDC-NH2 (SEQ IDNO:15) 0% (inhibition observed at 500 μg/mL) N-Ac-CRAHGVDC-NH2 (SEQ IDNO:58) 0% N-Ac-CLRAHAVC-NH2 (SEQ ID NO:59) 0% N-Ac-CLRAHGVC-NH2 (SEQ IDNO:60) 0% N-Ac-CLRAHAVDC-NH2 (SEQ ID NO:16) 60% N-Ac-CLRAHGVDC-NH2 (SEQID NO:61) 0% H-CAHAVDC-NH2 (SEQ ID NO:13) 0% H-CAHCVDC-NH2 (SEQ IDNO:62) 0% H-CAHAVDIC-NH2 (SEQ ID NO:14) 0% H-CAHGVDIC-NH2 (SEQ ID NO:57)0% E-cadherin specific N-Ac-CSHAVC-NH2 (SEQ ID NO:41) 0% N-Ac-CSHGVC-NH2(SEQ ID NO:63) 0% N-Ac-CHAVSC-NH2 (SEQ ID NO:42) 0% N-Ac-CHGVSC-NH2 (SEQID NO:64) 0% N-Ac-CSHAVSC-NH2 (SEQ ID NO:65) 0% N-Ac-CSHGVSC-NH2 (SEQ IDNO:66) 0% N-Ac-CSHAVSSC-NH2 (SEQ ID NO:43) 0% N-Ac-CSHGVSSC-NH2 (SEQ IDNO:67) 0% N-Ac-CHAVSSC-NH2 (SEQ ID NO:68) 0% N-Ac-CHGVSSC-NH2 (SEQ IDNO:69) 0%

EXAMPLE 4

Toxicity and Cell Proliferation Studies

This Example illustrates the initial work to evaluate the cytotoxiceffects of representative cyclic peptides.

N-Ac-CHAVC-NH₂ (SEQ ID NO:10) and the control peptide N-Ac-CHGVC-NH₂(SEQ ID NO:20) were evaluated for possible cytotoxic effects on humanmicrovascular endothelial (HMVEC; Clonetics), human umbilical veinendothelial (HUVEC; ATCC #CRL-1730), IAFp2 (human fibroblast cell line;Institute Armand-Frapier, Montreal, Quebec), WI-38 (human fibroblastcell line; ATCC #CCL-75), MDA-MB231 (human breast cancer cell line; ATCC#HTB-26), and PC-3 (human prostate cancer cell line; ATCC #CRL-1435)cells utilizing the MTT assay (Plumb et al., Cancer Res. 49:4435-4440,1989). Neither of the peptides was cytotoxic at concentrations up to andincluding 100 μM. Similarly, neither of the peptides was capable ofinhibiting the proliferation of the above cell lines at concentrationsup to 100 μM, as judged by ³H-thymidine incorporation assays.

In fact, none of the compounds tested thus far show any cytotoxicity atconcentrations up to and including 100 μM (Table 6 and 7). However,N-Ac-CHAVSC-NH₂ (SEQ ID NO:42), N-Ac-CHGVSC-NH₂ (SEQ ID NO:64),N-Ac-CVAHC-NH₂ (SEQ ID NO:70), N-Ac-CVGHC-NH₂ (SEQ ID NO: 71) andN-Ac-CSHAVSSC-NH₂ (SEQ ID NO:43) inhibited the proliferation of HUVEC atconcentrations (IC₅₀ values) of 57 μM, 42 μM, 8 μM, 30 μM and 69 μMrespectively, as judged by ³H-thymidine incorporation assays.N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:43) also inhibited the proliferation ofMDA-MB231 cells at a concentration of 76 μM and HMVEC cells at aconcentration of 70 μM (Tables 6 and 7). N-Ac-CHAVSC-NH₂ (SEQ ID NO:42)inhibited the proliferation of MDA-MB231 cells at a concentration of 52μM.

TABLE 6 Evaluation of Peptides for Cytotoxicity and Capacity to InhibitCell Proliferation of Normal Cells (IC₅O in μM) SEQ HMVEC HUVEC IAFp2WI-38 ID Cell Cell Cell Cell Peptide NO. prol Cytotox Prol Cytotox ProlCytotox Prol Cytot N-Ac-CHGVC-NH₂ 20 >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 (control for #1) N-Ac-CHAVC-NH₂ 10 >100μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 (#1) H-CHGVC-NH₂20 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 (controlfor #2) H-CHAVC-NH₂ (#2) 10 >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100 μM >100 N-Ac-CHGVSC-NH₂ 64 >100 μM >100 μM 42 μM >100 μM >100μM >100 μM >100 μM >100 (control for #18) N-Ac-CHAVSC-NH₂ 42 >100μM >100 μM 57 μM >100 μM >100 μM >100 μM >100 μM >100 (#18)N-Ac-CSHGVC-NH₂ 63 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100 (control for #16) N-Ac-CSHAVC-NH₂ 41 >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 μM >100 (#16) N-Ac-CAHGVDC- 62 >100μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 NH₂ (control for#22) N-Ac-CAHAVDC- 13 >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100 μM >100 NH₂ (#22) N-Ac-KHGVD-NH₂ 54 >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 (control for #26) N-Ac-KHAVD-NH₂ 53 >100μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 (#26)H-CAHGVDC-NH₂ 62 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100 (control for #45) H-CAHAVDC-NH₂ 13 >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 (#45) H-CAHGVDIC-NH₂ 57 >100 μM >100μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 (control for #47)H-CAHAVDIC-NH₂ 14 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100 (#47) N-Ac-CVGHC-NH₂ 71 >100 μM >100 μM 30 μM >100 μM >100μM >100 μM >100 μM >100 (control for #32) N-Ac-CVAHC-NH₂ 70 >100 μM >100μM 8 μM >100 μM >100 μM >100 μM >100 μM >100 (#32) N-Ac-CAHGVDIC-57 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 NH₂(control for #14) N-Ac-CAHAVDIC- 14 >100 μM >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100 NH₂ (#14) N-Ac-CSHGVSSC- 67 >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 μM >100 NH2 (control for #24)N-Ac-CSHAVSSC- 43 70 μM >100 μM 69 μM >100 μM >100 μM >100 μM >100μM >100 NH₂* (#24) *Incompletely soluble in RPMI at 1 mM

TABLE 7 Evaluation of Peptides for Cytotoxicity and Capacity to InhibitCell Proliferation of Tumoral Cells (IC₅₀ in μM) SEQ Tumoral Cells IDMDA-MB231 PC-3 Peptide NO: Cell Prol Cytotox Cell Prol CytotoxN-Ac-CHGVC-NH₂ (control 20 >100 μM >100 μM >100 μM >100 μM for #1)N-Ac-CHAVC-NH₂ (#1) 10 >100 μM >100 μM >100 μM >100 μM H-CHGVC-NH₂(control for 20 >100 μM >100 μM >100 μM >100 μM #2) H-CHAVC-NH₂ (#2)10 >100 μM >100 μM >100 μM >100 μM N-Ac-CHGVSC-NH₂ 64 >100 μM >100μM >100 μM >100 μM (control for #18) N-Ac-CHAVSC-NH₂* (#18) 42 52μM >100 μM >100 μM >100 μM N-Ac-CSHGVC-NH₂ 63 >100 μM >100 μM >100μM >100 μM (control for #6) N-Ac-CSHAVC-NH₂ (#16) 41 >100 μM >100μM >100 μM >100 μM N-Ac-CAHGVDC-NH₂ 62 >100 μM >100 μM >100 μM >100 μM(control for #22) N-Ac-CAHAVDC-NH₂ 13 >100 μM >100 μM >100 μM >100 μM(#22) N-Ac-KHGVD-NH₂ 54 >100 μM >100 μM >100 μM >100 μM (control for#26) N-Ac-KHAVD-NH₂ (#26) 53 >100 μM >100 μM >100 μM >100 μMH-CAHGVDC-NH₂ 62 >100 μM >100 μM >100 μM >100 μM (control for #45)H-CAHAVDC-NH₂ (#45) 13 >100 μM >100 μM >100 μM >100 μM H-CAHGVDIC-NH₂57 >100 μM >100 μM >100 μM >100 μM (control for #47) H-CAHAVDIC-NH₂(#47) 14 >100 μM >100 μM >100 μM >100 μM N-Ac-CVGHC-NH₂ 71 >100 μM >100μM >100 μM >100 μM (control for #32) N-Ac-CVAHC-NH₂ (#32) 70 >100μM >100 μM >100 μM >100 μM N-Ac-CAHGVDIC-NH₂ 57 >100 μM >100 μM >100μM >100 μM (control for #14) N-Ac-CAHAVDIC-NH₂ 14 >100 μM >100 μM >100μM >100 μM (#14) N-Ac-CSHGVSSC-NH₂ 67 >100 μM >100 μM >100 μM >100 μM(control for #24) N-Ac-CSHAVSSC-NH₂ * 43 76 μM >100 μM >100 μM >100 μM(#24) *Incompletely soluble in RPMI at 1 mM

EXAMPLE 5

Chronic Toxicity Study

This Example illustrates a toxicity study performed using arepresentative cyclic peptide.

Varying amounts of H-CHAVC-NH₂ (SEQ ID NO:10; 2 mg/kg, 20 mg/kg and 125mg/kg) were injected into mice intraperitoneally every day for threedays. During the recovery period (days 4-8), animals were observed forclinical symptoms. Body weight was measured (FIG. 8) and no significantdifferences occurred. In addition, no clinical symptoms were observed onthe treatment or recovery days. Following the four day recovery period,autopsies were performed and no abnormalities were observed.

EXAMPLE 6

Stability of Cyclic Peptide in Blood

This Example illustrates the stability of a representative cyclicpeptide in mouse whole blood.

50 μl of a stock solution containing 12.5 μg/ml H-CHAVC-NH₂ (SEQ IDNO:10) was added to mouse whole blood and incubated at 37° C. Aliquotswere removed at intervals up to 240 minutes, precipitated withacetonitrile, centrifuged and analyzed by HPLC. The results (Table 8 andFIG. 9) are expressed as % remaining at the various time points, andshow generally good stability in blood.

TABLE 8 Stability of Representative Cyclic Peptide in Mouse Whole BloodTime (Min.) Area 1 Area 2 Average % Remaining  0 341344 246905  294124.5 100.00 10 308924 273072 290998 98.94 20 289861 220056  254958.5 86.68 30 353019 310559 331789 112.81 45 376231 270860  323545.5 110.00 60 373695 188255 280975 95.53 90 435555 216709 326132110.88 120  231694 168880 200287 68.10 240  221952 242148 232050 78.90

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention.

77 1 108 PRT Homo sapiens 1 Asp Trp Val Ile Pro Pro Ile Asn Leu Pro GluAsn Ser Arg Gly Pro 1 5 10 15 Phe Pro Gln Glu Leu Val Arg Ile Arg SerAsp Arg Asp Lys Asn Leu 20 25 30 Ser Leu Arg Tyr Ser Val Thr Gly Pro GlyAla Asp Gln Pro Pro Thr 35 40 45 Gly Ile Phe Ile Leu Asn Pro Ile Ser GlyGln Leu Ser Val Thr Lys 50 55 60 Pro Leu Asp Arg Glu Gln Ile Ala Arg PheHis Leu Arg Ala His Ala 65 70 75 80 Val Asp Ile Asn Gly Asn Gln Val GluAsn Pro Ile Asp Ile Val Ile 85 90 95 Asn Val Ile Asp Met Asn Asp Asn ArgPro Glu Phe 100 105 2 108 PRT Mus musculus 2 Asp Trp Val Ile Pro Pro IleAsn Leu Pro Glu Asn Ser Arg Gly Pro 1 5 10 15 Phe Pro Gln Glu Leu ValArg Ile Arg Ser Asp Arg Asp Lys Asn Leu 20 25 30 Ser Leu Arg Tyr Ser ValThr Gly Pro Gly Ala Asp Gln Pro Pro Thr 35 40 45 Gly Ile Phe Ile Ile AsnPro Ile Ser Gly Gln Leu Ser Val Thr Lys 50 55 60 Pro Leu Asp Arg Glu LeuIle Ala Arg Phe His Leu Arg Ala His Ala 65 70 75 80 Val Asp Ile Asn GlyAsn Gln Val Glu Asn Pro Ile Asp Ile Val Ile 85 90 95 Asn Val Ile Asp MetAsn Asp Asn Arg Pro Glu Phe 100 105 3 108 PRT Bos taurus 3 Asp Trp ValIle Pro Pro Ile Asn Leu Pro Glu Asn Ser Arg Gly Pro 1 5 10 15 Phe ProGln Glu Leu Val Arg Ile Arg Ser Asp Arg Asp Lys Asn Leu 20 25 30 Ser LeuArg Tyr Ser Val Thr Gly Pro Gly Ala Asp Gln Pro Pro Thr 35 40 45 Gly IlePhe Ile Ile Asn Pro Ile Ser Gly Gln Leu Ser Val Thr Lys 50 55 60 Pro LeuAsp Arg Glu Leu Ile Ala Arg Phe His Leu Arg Ala His Ala 65 70 75 80 ValAsp Ile Asn Gly Asn Gln Val Glu Asn Pro Ile Asp Ile Val Ile 85 90 95 AsnVal Ile Asp Met Asn Asp Asn Arg Pro Glu Phe 100 105 4 108 PRT Homosapiens 4 Asp Trp Val Val Ala Pro Ile Ser Val Pro Glu Asn Gly Lys GlyPro 1 5 10 15 Phe Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp ArgAsp Thr 20 25 30 Lys Ile Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser ProPro Glu 35 40 45 Gly Val Phe Ala Val Glu Lys Glu Thr Gly Trp Leu Leu LeuAsn Lys 50 55 60 Pro Leu Asp Arg Glu Glu Ile Ala Lys Tyr Glu Leu Phe GlyHis Ala 65 70 75 80 Val Ser Glu Asn Gly Ala Ser Val Glu Asp Pro Met AsnIle Ser Ile 85 90 95 Ile Val Thr Asp Gln Asn Asp His Lys Pro Lys Phe 100105 5 108 PRT Mus musculus 5 Glu Trp Val Met Pro Pro Ile Phe Val Pro GluAsn Gly Lys Gly Pro 1 5 10 15 Phe Pro Gln Arg Leu Asn Gln Leu Lys SerAsn Lys Asp Arg Gly Thr 20 25 30 Lys Ile Phe Tyr Ser Ile Thr Gly Pro GlyAla Asp Ser Pro Pro Glu 35 40 45 Gly Val Phe Thr Ile Glu Lys Glu Ser GlyTrp Leu Leu Leu His Met 50 55 60 Pro Leu Asp Arg Glu Lys Ile Val Lys TyrGlu Leu Tyr Gly His Ala 65 70 75 80 Val Ser Glu Asn Gly Ala Ser Val GluGlu Pro Met Asn Ile Ser Ile 85 90 95 Ile Val Thr Asp Gln Asn Asp Asn LysPro Lys Phe 100 105 6 108 PRT Homo sapiens 6 Asp Trp Val Ile Pro Pro IleSer Cys Pro Glu Asn Glu Lys Gly Pro 1 5 10 15 Phe Pro Lys Asn Leu ValGln Ile Lys Ser Asn Lys Asp Lys Glu Gly 20 25 30 Lys Val Phe Tyr Ser IleThr Gly Gln Gly Ala Asp Thr Pro Pro Val 35 40 45 Gly Val Phe Ile Ile GluArg Glu Thr Gly Trp Leu Lys Val Thr Glu 50 55 60 Pro Leu Asp Arg Glu ArgIle Ala Thr Tyr Thr Leu Phe Ser His Ala 65 70 75 80 Val Ser Ser Asn GlyAsn Ala Val Glu Asp Pro Met Glu Ile Leu Ile 85 90 95 Thr Val Thr Asp GlnAsn Asp Asn Lys Pro Glu Phe 100 105 7 108 PRT Mus musculus 7 Asp Trp ValIle Pro Pro Ile Ser Cys Pro Glu Asn Glu Lys Gly Glu 1 5 10 15 Phe ProLys Asn Leu Val Gln Ile Lys Ser Asn Arg Asp Lys Glu Thr 20 25 30 Lys ValPhe Tyr Ser Ile Thr Gly Gln Gly Ala Asp Lys Pro Pro Val 35 40 45 Gly ValPhe Ile Ile Glu Arg Glu Thr Gly Trp Leu Lys Val Thr Gln 50 55 60 Pro LeuAsp Arg Glu Ala Ile Ala Lys Tyr Ile Leu Tyr Ser His Ala 65 70 75 80 ValSer Ser Asn Gly Glu Ala Val Glu Asp Pro Met Glu Ile Val Ile 85 90 95 ThrVal Thr Asp Gln Asn Asp Asn Arg Pro Glu Phe 100 105 8 5 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 8 AspXaa Asn Asp Asn 1 5 9 4 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 9 Leu Asp Arg Glu 1 10 5 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 10 Cys His Ala Val Cys 1 5 11 6 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 11 Cys His AlaVal Asp Cys 1 5 12 6 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 12 Cys Ala His Ala Val Cys 1 5 13 7 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 13 Cys Ala His Ala Val Asp Cys 1 5 14 8 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 14 CysAla His Ala Val Asp Ile Cys 1 5 15 8 PRT Artificial Sequence Descriptionof Artificial Sequence Solid Phase Synthesis 15 Cys Arg Ala His Ala ValAsp Cys 1 5 16 9 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 16 Cys Leu Arg Ala His Ala Val Asp Cys 15 17 5 PRT Artificial Sequence Description of Artificial Sequence SolidPhase Synthesis 17 Asp His Ala Val Lys 1 5 18 5 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 18 Lys His AlaVal Glu 1 5 19 6 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 19 Ala His Ala Val Asp Ile 1 5 20 5 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 20 Cys His Gly Val Cys 1 5 21 5 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 21 Tyr Ile GlySer Arg 1 5 22 10 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 22 Lys Tyr Ser Phe Asn Tyr Asp Gly SerGlu 1 5 10 23 17 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 23 Ile Trp Lys His Lys Gly Arg Asp ValIle Leu Lys Lys Asp Val Arg 1 5 10 15 Phe 24 4 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 24 Ile Asp AspLys 1 25 4 PRT Artificial Sequence Description of Artificial SequenceSolid Phase Synthesis 25 Asp Asp Lys Ser 1 26 5 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 26 Val Ile AspAsp Lys 1 5 27 5 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 27 Ile Asp Asp Lys Ser 1 5 28 6 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 28 Val Ile Asp Asp Lys Ser 1 5 29 5 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 29 Asp Asp LysSer Gly 1 5 30 6 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 30 Ile Asp Asp Lys Ser Gly 1 5 31 7 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 31 Val Ile Asp Asp Lys Ser Gly 1 5 32 6 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 32 PheVal Ile Asp Asp Lys 1 5 33 7 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 33 Phe Val Ile Asp Asp Lys Ser1 5 34 8 PRT Artificial Sequence Description of Artificial SequenceSolid Phase Synthesis 34 Phe Val Ile Asp Asp Lys Ser Gly 1 5 35 7 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 35 Ile Phe Val Ile Asp Asp Lys 1 5 36 8 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 36 IlePhe Val Ile Asp Asp Lys Ser 1 5 37 9 PRT Artificial Sequence Descriptionof Artificial Sequence Solid Phase Synthesis 37 Ile Phe Val Ile Asp AspLys Ser Gly 1 5 38 10 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 38 Cys Asp Gly Tyr Pro Lys Asp Cys LysGly 1 5 10 39 10 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 39 Cys Gly Asn Leu Ser Thr Cys Met LeuGly 1 5 10 40 9 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 40 Cys Tyr Ile Gln Asn Cys Pro Leu Gly 15 41 6 PRT Artificial Sequence Description of Artificial Sequence SolidPhase Synthesis 41 Cys Ser His Ala Val Cys 1 5 42 6 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 42 CysHis Ala Val Ser Cys 1 5 43 8 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 43 Cys Ser His Ala Val Ser SerCys 1 5 44 5 PRT Artificial Sequence Description of Artificial SequenceSolid Phase Synthesis 44 Cys His Ala Val Xaa 1 5 45 10 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 45 IleXaa Tyr Ser His Ala Val Ser Cys Glu 1 5 10 46 10 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 46 Ile Xaa TyrSer His Ala Val Ser Ser Cys 1 5 10 47 9 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 47 Xaa Tyr SerHis Ala Val Ser Ser Cys 1 5 48 9 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 48 Xaa Tyr Ser His Ala Val SerSer Cys 1 5 49 6 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 49 Ser His Ala Val Ser Ser 1 5 50 5 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 50 His Ala Val Xaa Ser 1 5 51 4 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 51 Trp Gly GlyTrp 1 52 15 PRT Artificial Sequence Description of Artificial SequenceSolid Phase Synthesis 52 Phe His Leu Arg Ala His Ala Val Asp Ile Asn GlyAsn Gln Val 1 5 10 15 53 5 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 53 Lys His Ala Val Asp 1 5 545 PRT Artificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 54 Lys His Gly Val Asp 1 5 55 6 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 55 Cys His GlyVal Asp Cys 1 5 56 6 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 56 Cys Ala His Gly Val Cys 1 5 57 8 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 57 Cys Ala His Gly Val Asp Ile Cys 1 5 58 8 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 58 CysArg Ala His Gly Val Asp Cys 1 5 59 8 PRT Artificial Sequence Descriptionof Artificial Sequence Solid Phase Synthesis 59 Cys Leu Arg Ala His AlaVal Cys 1 5 60 8 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 60 Cys Leu Arg Ala His Gly Val Cys 1 5 619 PRT Artificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 61 Cys Leu Arg Ala His Gly Val Asp Cys 1 5 62 7 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 62 CysAla His Gly Val Asp Cys 1 5 63 6 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 63 Cys Ser His Gly Val Cys 1 564 6 PRT Artificial Sequence Description of Artificial Sequence SolidPhase Synthesis 64 Cys His Gly Val Ser Cys 1 5 65 7 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 65 CysSer His Ala Val Ser Cys 1 5 66 7 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 66 Cys Ser His Gly Val Ser Cys1 5 67 8 PRT Artificial Sequence Description of Artificial SequenceSolid Phase Synthesis 67 Cys Ser His Gly Val Ser Ser Cys 1 5 68 7 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 68 Cys His Ala Val Ser Ser Cys 1 5 69 7 PRT ArtificialSequence Description of Artificial Sequence Solid Phase Synthesis 69 CysHis Gly Val Ser Ser Cys 1 5 70 5 PRT Artificial Sequence Description ofArtificial Sequence Solid Phase Synthesis 70 Cys Val Ala His Cys 1 5 715 PRT Artificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 71 Cys Val Gly His Cys 1 5 72 5 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 72 Asp His GlyVal Lys 1 5 73 5 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 73 Lys His Gly Val Glu 1 5 74 6 PRTArtificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 74 Ala His Gly Val Asp Ile 1 5 75 6 PRT Artificial SequenceDescription of Artificial Sequence Solid Phase Synthesis 75 Ser His GlyVal Ser Ser 1 5 76 8 PRT Artificial Sequence Description of ArtificialSequence Solid Phase Synthesis 76 Lys Ser His Ala Val Ser Ser Asp 1 5 778 PRT Artificial Sequence Description of Artificial Sequence Solid PhaseSynthesis 77 Lys Ser His Gly Val Ser Ser Asp 1 5

What is claimed is:
 1. A method for modulating neurite outgrowth,comprising contacting a neuron with a cell adhesion modulating peptidethat comprises a cyclic peptide in which nonadjacent amino acid residuesare covalently linked to form a peptide ring, wherein the peptide ringcomprises the sequence His-Ala-Val within a cyclic peptide ring thatcontains 4-15 amino acid residues for a time under conditions to effectneurite outgrowth.
 2. A method according to claim 1, wherein the cyclicpeptide has the formula:

wherein X₁, and X₂ are optional, and if present, are independentlyselected from the group consisting of amino acid residues andcombinations thereof in which the residues are linked by peptide bonds,and wherein X₁ and X₂ independently range in size from 0 to 10 residues,such that the sum of residues contained within X₁ and X₂ ranges from 1to 12; wherein Y₁ and Y₂ are independently selected from the groupconsisting of amino acid residues, and wherein a covalent bond is formedbetween residues Y₁ and Y₂; and wherein Z₁ and Z₂ are optional, and ifpresent, are independently selected from the group consisting of aminoacid residues and combinations thereof in which the residues are linkedby peptide bonds.
 3. A method according to claim 2, wherein Z₁ is notpresent and Y₁ comprises an N-acetyl group.
 4. A method according toclaim 2, wherein Z₂ is not present and Y₂ comprises a C-terminal amidegroup.
 5. A method according to claim 2, wherein Y₁ and Y₂ arecovalently linked via a disulfide bond.
 6. A method according to claim5, wherein Y₁ and Y₂ are each independently selected from the groupconsisting of penicillamine, β,β-tetramethylene cysteine,β,β-pentamethylene cysteine, β-mercaptopropionic acid,β,β-pentamethylene-β-mercaptopropionic acid, 2 mercaptobenzene,2-mercaptoaniline, 2-mercaptoproline and derivatives thereof.
 7. Amethod according to claim 5, wherein Y₁ and Y₂ are cysteine residues orderivatives thereof.
 8. A method according to claim 2, wherein Y₁ and Y₂are covalently linked via an amide bond.
 9. A method according to claim8, wherein the amide bond is formed is formed between terminalfunctional groups.
 10. A method according to claim 8, wherein the amidebond is formed between residue side-chains.
 11. A method according toclaim 8, wherein the amide bond is formed between one terminalfunctional group and one residue side chain.
 12. A method according toclaim 8, wherein: (a) Y₁ is selected from the group consisting oflysine, ornithine, and derivatives thereof and Y₂ is selected from thegroup consisting of aspartate, glutamate and derivatives thereof; or (b)Y₂ is selected from the group consisting of lysine, ornithine andderivatives thereof and Y₁ is selected from the group consisting ofaspartate, glutamate and derivatives thereof.
 13. A method according toclaim 2, wherein Y₁ and Y₂ are covalently linked via a thioether bond.14. A method according to claim 2, wherein Y₁ and Y₂ are each tryptophanor a derivative thereof, such that the covalent bond generates aδ₁δ₁-ditryptophan, or a derivative thereof.
 15. A method according toclaim 1, wherein the modulating agent comprises a sequence selected fromthe group consisting of CHAVC (SEQ ID NO:10), CHAVDC (SEQ ID NO:11),CAHAVC (SEQ ID NO:12), CAHAVDC (SEQ ID NO:13), CAHAVDIC (SEQ ID NO:14),CRAHAVDC (SEQ ID NO:15), CLRAHAVDC (SEQ ID NO:16), DHAVK (SEQ ID NO:17),KHAVE (SEQ ID NO:18), AHAVDI (SEQ ID NO:19) and derivatives of theforegoing sequences having one or more C-terminal, N-terminal and/orside chain modifications.
 16. A method according to claim 1, whereinneurite outgrowth is inhibited.
 17. A method according to claim 1,wherein neurite outgrowth is enhanced.
 18. A method according to claim1, wherein neurite outgrowth is directed.
 19. A method according toclaim 1, wherein the modulating agent comprises at least two HAVsequences separated by a linker.
 20. A method, rding to claim 1, whereinthe modulating agent is linked to a drug.
 21. A method according toclaim 1, wherein the modulating agent is linked to a targeting agent.22. A method according to claim 1, wherein neurite outgrowth is enhancedand/or directed and wherein the modulating agent is linked to a solidsupport.
 23. A method according to claim 22, wherein the solid supportis a polymeric matrix.
 24. A method according to claim 23, wherein thesolid support is selected from the group consisting of plastic dishes,plastic tubes, sutures, membranes, ultra thin films, bioreactors andmicroparticles.
 25. A method according to claim 1, wherein themodulating agent is present within a pharmaceutical composition thatcomprises a pharmaceutically acceptable carrier.
 26. A method accordingto claim 25, wherein the composition further comprises a drug.
 27. Amethod according to claim 25, wherein the cell adhesion modulating agentis present within a sustained-release formulation.